3505 The Journal of Experimental Biology 213, 3505-3514 © 2010. Published by The Company of Biologists Ltd doi:10.1242/jeb.046110
Evolution of supercontraction in spider silk: structure–function relationship from tarantulas to orb-weavers Cecilia Boutry* and Todd Alan Blackledge Department of Biology and Integrated Bioscience Program, University of Akron, Akron, OH 44325-3908, USA *Author for correspondence (
[email protected])
Accepted 22 July 2010
SUMMARY Spider silk is a promising biomaterial with impressive performance. However, some spider silks also ‘supercontract’ when exposed to water, shrinking by up to ~50% in length. Supercontraction may provide a critical mechanism to tailor silk properties, both for future synthetic silk production and by the spiders themselves. Several hypotheses are proposed for the mechanism and function of supercontraction, but they remain largely untested. In particular, supercontraction may result from a rearrangement of the GPGXX motif within the silk proteins, where G represents glycine, P proline and X is one of a small subset of amino acids. Supercontraction may prevent sagging in wet orb-webs or allow spiders to tailor silk properties for different ecological functions. Because both the molecular structures of silk proteins and how dragline is used in webs differ among species, we can test these hypotheses by comparing supercontraction of silk across diverse spider taxa. In this study we measured supercontraction in 28 spider taxa, ranging from tarantulas to orb-weaving spiders. We found that silk from all species supercontracted, except that of most tarantulas. This suggests that supercontraction evolved at least with the origin of the Araneomorphae, over 200 million years ago. We found differences in the pattern of evolution for two components of supercontraction. Stress generated during supercontraction of a restrained fiber is not associated with changes in silk structure and web architecture. By contrast, the shrink of unrestrained supercontracting fibers is higher for Orbiculariae spiders, whose silk contains high ratios of GPGXX motifs. These results support the hypothesis that supercontraction is caused by a rearrangement of GPGXX motifs in silk, and that it functions to tailor silk material properties. Key words: spider silk, supercontraction, biomaterials, biomechanics.
INTRODUCTION
Spider major ampullate silk is a promising biomaterial, combining high strength and elasticity (Gosline et al., 1986). Furthermore, silk is biocompatible (Allmeling et al., 2006; Gellynck et al., 2006). Potential applications range from artificial tendons and ligaments (Kluge et al., 2008) to microspheres for drug delivery (Lammel et al., 2008). However, large amounts of spider silk are hard to obtain. Researchers are therefore working to produce synthetics fibers based on spider major ampullate silk (Vendrely and Scheibel, 2007). However, in contrast to most known materials, silk supercontracts under mild conditions (when humidity rises above ~70%) (Work, 1977). During supercontraction, water infiltrates the silk and causes it to shrink, up to half its dry length (Work, 1977).This process also generates high stresses if the fiber is restrained. Supercontraction could play a critical role in the production of dragline silk by spiders by allowing spiders to ‘tailor’ silk properties (Guinea et al., 2005a). Although it can hinder certain applications of silk, it can also lead to new uses that involve silk moving objects rather than simply resisting loads (Agnarsson et al., 2009b). Thus, there is a crucial need to understand the mechanisms of supercontraction. Supercontraction is relatively well documented among orbweaving spiders such as Araneidae and Nephilidae (Grubb and Ji, 1999; Savage et al., 2004; van Beek et al., 2002; Work, 1981), and was also found in the Pisauridae (Shao and Vollrath, 1999) and Theridiidae (Shao and Vollrath, 1999; Work, 1981). Whether silk from other taxa supercontracts, in particular silk from ‘basal’ taxa such as tarantulas and haplogynes (e.g. daddy long leg and spitting spiders), remains uninvestigated. The current molecular model for
supercontraction (Eles and Michal, 2004; Termonia, 1994) and the possible functions proposed for supercontraction (Guinea et al., 2003; Guinea et al., 2005a; Lewis, 1992; Work, 1981) are largely based on our knowledge of silk composition and web ecology of members of the Araneidae and Nephilidae, a small fraction of all existing spiders (~10% of spiders species). Understanding the supercontraction behavior of silk from other taxa, with different ecologies and silk composition, provides a crucial test of the proposed mechanisms and functions of supercontraction. Here, we present the first comprehensive study of supercontraction in a wide range of spiders and use a phylogenetic perspective to understand the origin and function of supercontraction in spider major ampullate silk. Spider major ampullate silk is composed of proteins containing repeated amino acid motifs, i.e. short, stereotyped amino acid sequences that form specific secondary structures. The major ampullate silk of the Orbiculariae contains poly-alanine and glycine–alanine motifs that form -sheet crystals (Jelinski et al., 1999; Kümmerlen et al., 1996; Simmons et al., 1994; Xu and Lewis, 1990), glycine–glycine–X motifs that form 310 helices (Bram et al., 1997; van Beek et al., 2002) and glycine–proline–glycine motifs (Ayoub et al., 2007; Hayashi and Lewis, 1998; Hayashi et al., 1999; Hinman et al., 2000; Hinman and Lewis, 1992). There is no consensus as to what structures are formed by the glycine–proline– glycine motifs. They have been described as helical fractions (Vollrath and Porter, 2009), proline-rich network chains (Savage and Gosline, 2008a), -spirals (Hayashi and Lewis, 1998; Hayashi et al., 1999) and various types of -turns (Ohgo et al., 2006). In
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3506 C. Boutry and T. A. Blackledge this paper, we will simply refer to these motifs as GPGXX motifs, where G represents glycine, P proline and X any one of a small subset of amino acids. Currently, supercontraction is hypothesized to result from rearrangements of the non-crystalline fractions formed by the GPGXX motifs and the 310 helices within the silk fiber (Blackledge et al., 2009a; Eles and Michal, 2004; Termonia, 1994). When the silk is dry, these non-crystalline regions are maintained parallel to the fiber axis by hydrogen bonds. However, when the humidity rises, water disrupts these hydrogen bonds, allowing the non-crystalline regions to rearrange to lower energetic configurations, driving supercontraction (Eles and Michal, 2004; Savage and Gosline, 2008b; Yang et al., 2000). This rearrangement leads to the shrinking and thickening of the fiber and, at the molecular level, to an observed loss of orientation (Grubb and Ji, 1999; Parkhe et al., 1997). If supercontraction is induced by a rearrangement of the glycine– glycine–X or GPGGXX motifs, then there should be a positive relationship between abundance of these motifs in the silk and strength of supercontraction. Major ampullate silk contains one or two types of proteins, both termed major ampullate spidroins or MaSp for short (Hinman and Lewis, 1992; Xu and Lewis, 1990). Mygalomorphs (tarantulas) lack clearly differentiated silk glands (Palmer, 1985; Palmer et al., 1982). Their silk proteins contain long repeats, rich in alanine and serine (Garb et al., 2007). Major ampullate glands appeared with the Araneomorphae spiders, which include haplogyne spiders such as daddy long leg spiders, and entelegyne spiders. Haplogyne major ampullate silk is composed of long repeat units rich in alanine, serine and glycine (Gatesy et al., 2001). These proteins differ from the major ampullate spidroins found in the sister taxon to the haplogyne, the entelegyne spiders, which include most common spiders, such as orb-weavers and wolf spiders. Entelegynes possess a MaSp1-like protein, rich in polyalanine and glycine–alanine repeats that form -sheets, as well as glycine–glycine–X helices (Gatesy et al., 2001; PouchkinaStantcheva and McQueen-Mason, 2004) (but see Tian et al., 2004). The second protein, MaSp2, includes GPGXX motifs (Hinman and Lewis, 1992) acting as molecular nanosprings (Becker et al., 2003). MaSp2 is known to be produced by the Orbiculariae (orb-weaving spiders and their relatives) but is probably absent from all other taxa (see Materials and methods). Therefore, if supercontraction results from the rearrangement of GPGXX motifs, silk containing MaSp2 proteins (i.e. Orbiculariae silk) should supercontract more than silk lacking MaSp2. Such a phylogenetically based approach may also provide insight into the two functions proposed for supercontraction: tailoring of silk properties during fiber spinning and tightening of orb webs loaded with water. According to the tailoring hypothesis, silk is in a supercontracted state at the beginning of the spinning process, when it is first drawn from a liquid solution. The extent to which the supercontracted silk is stretched during spinning determines molecular alignment, and thereby, the properties of the fiber after extrusion and drying (Guinea et al., 2005a). The tailoring hypothesis predicts that supercontraction was selected for in spiders that use major ampullate silk in diverse ecological contexts. For instance, members of basal spider taxa, such as tarantulas, largely use sheets of silk to line burrows or to construct ‘simple’ brushed sheet webs on the substrate. Discrete major ampullate silk threads are first used in webs of haplogyne spiders. However, their webs tend to be relatively simple and constructed close to the substrate. Examples include the ‘lampshade’ web of Hypochilus and the disorganized sheet webs of Kukulcania. Entelegynes, the sister taxa of haplogynes, include, among others,
two clades that dramatically shifted how they use dragline silk. Most RTA (retrolateral tibial apophysis) clade species, such as jumping spiders and wolf spiders, do not spin capture webs and only lay a trail of dragline silk as they walk. By contrast, members of the Orbiculariae not only use draglines, but also spin a diversity of complex webs composed of distinct architectural elements, such as orb-webs and cobwebs. These webs are suspended in the air and have multiple discrete elements made of major ampullate silk (e.g. radii, frame and mooring guys in orb-webs). These elements serve distinct functions that place different demands on the threads in terms of mechanical performance. Orbicularian spiders may thus need to spin silk threads with different material properties depending on the threads’ function. Therefore, the ability to tailor silk properties may have been selected for in the Orbiculariae. By contrast, species that do not use silk in webs (many tarantulas and RTA clade spiders) may have less need to modulate silk properties. If tailoring of silk is achieved through supercontraction (Guinea et al., 2005a), then higher supercontraction shrink and stress should have been selected for in Orbiculariae compared with other taxa. The second hypothesized function of supercontraction is to prevent orb-webs from sagging under the weight of dew drops by tensing threads (Guinea et al., 2003; Lewis, 1992; Work, 1981). This hypothesis predicts that supercontraction has been selected for in species that spin aerial orb-webs in contrast to non-orb-weaving species. Orb-webs are spun only by members of the Orbiculariae. Furthermore, several derived families of Orbiculariae spin different web types, such as the cobwebs of the Theridiidae (Coddington and Levi, 1991; Eberhard et al., 2008). Planar orb webs contain major ampullate radii that only are in contact at the center of the web. By contrast, in cobwebs, each major ampullate support thread contacts many other threads, forming a complex, seemingly disorganized, network. Since cobweb threads connects with many other threads, loads may be better distributed between threads than they are in orb webs, which may allow cobwebs to resist loads better than orbwebs. Therefore, unlike orb-webs, cobwebs may not need high tension to resist the load of dew drops. Hence, if supercontraction has been selected for web tightening, supercontraction may have been secondarily lost in the Orbiculariae that lost the orb-web. To summarize, if supercontraction is caused by GPGXX motifs, then all spiders producing silk rich in MaSp2 should spin major ampullate silk that supercontracts more, so that all Orbiculariae should exhibit higher supercontraction than all other taxa. The same pattern is predicted if supercontraction evolved under selection for tailoring silk properties. By contrast, if supercontraction functions to tighten wet orb-webs, then orb-weaving species within the Orbiculariae should spin silk that supercontracts more than non-orb-weaving species, whether these are Orbiculariae or not (Table1). Under this hypothesis, we predict that non-orb-weaving Orbiculariae lost supercontraction as they switched to three-dimensional webs because supercontraction did not yield any advantage for web protection from water drops in these species, thereby relaxing selection for it. Table 1. Predicted levels of supercontraction for different spider taxa as a function of the proposed hypotheses on supercontraction mechanisms and function Orbiculariae
Mechanism: GPGXX motifs Function: silk tailoring Function: web tightening
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Orb-web spinners
Non-orb spinners
Other spiders
+ + +
+ + –
– – –
Evolution of silk supercontraction
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Table 2. Taxa used in this study with indications of the spidroins present in the silk and the type of web Species
Ephebopus uatuman (Lucas et al.) Grammostola rosea (Walckenaer) Aphonopelma seemani (F.O.P. Cambridge) Hypochilus thorelli (Marx) Kukulcania hibernalis (Hentz) Diguetia canities (McCook) Pholcus phalangioides (Fuesslin) Scytodes sp. (Latreille) Eresus kollari (Rossi) Hololena adnexa (Chamberlin and Gertsch) Hogna helluo (Walckenaer) Amaurobius ferox (Walckenaer) Salticus scenicus (Clerck) Tengella radiata (Kulczynski) Dolomedes tenebrosus (Hentz) Peucetia viridans (Hentz) Uloborus diversus (Marx) Pityohyphantes costatus (Hentz) Tetragnatha sp. (Latreille) Latrodectus hesperus (Chamberlin and Ivie) Achaearanea tepidariorum (Koch) Synotaxus sp. (Simon) Nephila clavipes (Linnaeus) Zygiella x-notata (Clerck) Araneus diadematus (Clerck) Verrucosa arenata (Walckenaer) Larinioides sclopetarius (Clerck) Nuctenea umbratica (Clerck)
Family
MaSp2 present
Web type
Silk collection
Origin
No. spiders; sample*
Theraphosidae Theraphosidae Theraphosidae Hypochilidae Filistatidae Diguetidae Pholcidae Scytodidae Eresidae Agelenidae Lycosidae Amaurobiidae Salticidae Tengellidae Pisauridae Oxyopidae Uloboridae Linyphiidae Tetragnathidae Theridiidae Theridiidae Theridiidae Nephilidae Araneidae Araneidae Araneidae Araneidae Araneidae
No (I) No (I) No (I) No (I) No No (I) No (I) No (I) No (I) No No (I) No (I) No (I) No (I) No No (I) Yes Yes (I) Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes
No web No web No web Lampshade Sheet Tentweb Tangle No web Tube Funnel No web Funnel No web Funnel No web No web Orb Sheet Orb Cobweb Cobweb “Mesh” Orb Orb Orb Orb Orb Orb
NS NS NS NS NS FS FS NS NS FS FS FS FS FS FS FS FS FS FS FS FS FS FS FS FS FS FS FS
TarantulaSpiders.com TarantulaSpiders.com TarantulaSpiders.com USA, TN SpiderPharm SpiderPharm Czech Republic Costa Rica Czech Republic USA, CA USA, OH USA, VA USA, OH Costa Rica USA, OH SpiderPharm USA, CA USA, OH USA, OH USA, CA USA, OH Costa Rica USA, FL Slovenia USA, OH USA, OH USA, OH Slovenia
3; 8 5; 13 2; 4 4; 19 7; 34 2; 7 4; 8 4; 29 3; 7 5; 16 2; 16 3; 12 2; 7 4; 14 2; 7 8; 36 8; 30 2; 7 2; 8 9; 44 9; 34 2; 8 6; 24 8; 32 3; 12 2; 15 4; 19 6; 22
If the presence of MaSp2 had not been investigated in the spider’s family and we inferred presence or absence of MaSp2 from the phylogeny, ‘(I)’ was added in column 3. FS, forcible silking; NS, naturally spun. *The last column indicates the number of individual spiders per species and the total number of silk samples tested in this study.
However, it is also possible that supercontraction was somewhat maintained as it is associated with a desirable property of silk. We tested these hypotheses by investigating supercontraction in 28 species from 21 families of the order Araneae. Finally, we examined two different aspects of supercontraction across spiders. Unrestrained fibers shrink as they contract whereas restrained fibers instead develop tension. These two aspects of supercontraction may have evolved under different selective forces. By measuring supercontraction in many diverse taxa, this study can begin to separate the different evolutionary pressures that shaped both aspects of supercontraction. MATERIALS AND METHODS Spider maintenance and silk collection
Most spiders were wild caught but some were purchased from either SpiderPharm (Yarnell, AZ, USA) or TarantulaSpiders.com (FL, USA). Spiders were housed in a variety of cages, depending upon their web spinning behaviors, and maintained in the laboratory at 24°C under a 15h:9h light:dark cycle. Spiders were silked within a week after entering the laboratory. Table2 presents the taxa used in this study, as well as their origin, silk collection method and numbers of individuals and thread samples used. Silk was mainly collected using forcible silking. The spider was anesthetized with carbon dioxide and taped down, ventral side facing up, on a Petri dish. Major ampullate silk was manually reeled off the spinnerets at ~10cms–1, and collected on cut-out cards across 15.3mm gaps. The silk was glued on either side of the gap using cyanoacrylate glue (Superglue®) (Blackledge et al., 2005b). During the process, the spinnerets and silk threads were observed under a stereomicroscope, to ensure the silk collected came from the major
ampullate spigot. Three to four samples were collected and tested for each individual spider. For a few taxa, it was impossible to collect silk by forcible silking. In this case, naturally spun silk was collected. The spider was allowed to run across a fan-shaped piece of cardboard. As it ran, the spider laid a trail of dragline silk across the peaks of the cardboard, which was collected onto cut-out cards. As with forcibly obtained silk, the threads were glued on each side of the 15.3mm gap with cyanoacrylate glue, and three to four samples were collected per spider. Dragline silk is composed of major ampullate silk strands, sometimes accompanied by thinner minor ampullate silk strands. The samples were observed under a microscope, and all samples that contained thin, minor ampullate, strands were discarded. Thus, the samples we used were made of one or two strands of major ampullate silk only. Naturally spun silk tends to be more compliant and weaker than forcibly-obtained silk, probably because of its decreased molecular orientation (Guinea et al., 2005b; Madsen and Vollrath, 1999; PerezRigueiro et al., 2001). However, the silks that were naturally spun did not drastically differ in their supercontraction behavior from the silks that were forcibly-obtained from related species (see below). Therefore, we think that differences in collection methods per se had only minor effects on our results. Tarantulas lack well-differentiated silk glands and therefore do not produce major ampullate silk. However, tarantulas use their silk for functions analogous to major ampullate silk, such as lining burrows. Furthermore, as tarantulas belong to the Mygalomorphae, the sister group to the clade of spiders producing major ampullate silk, their silk is ideal for an outgroup comparison.
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3508 C. Boutry and T. A. Blackledge Silk diameter measurements and supercontraction tests
SS F/A ,
(1)
where F is the force generated by the sample and A is the area of the sample. The fiber was then relaxed to half its original length (l0) so that it was completely slacked, and immediately pulled at 0.01mms–1 to twice its original length, while the load was recorded. If the fiber had been unrestrained when the humidity was increased, it would have shrunk from l0 to a postsupercontraction length l1. When the slacked fibers were stretched to this post-supercontraction length l1, a stress developed within the fiber. This allowed us to measure l1. Percentage of shrink (PS), which is the proportion by which the fiber shrinks when supercontracting, was then calculated as: PS (l0 – l1) / l0 .
(2)
For certain silks, fibers were still under tension after relaxing to half their original length. In these cases, the fibers were relaxed further. The samples that still presented a stress before the beginning of the pull were discarded. Correlation between SS, PS and preload tension
Samples with a high preload tension, that is samples with a high tension within the sample prior to supercontraction, exhibited no SS even though they supercontracted, as evidenced by their positive PS. This suggested that preload tension influenced SS. This was a particularly important issue because supercontraction tests were performed at constant 0.1% strain, which could result in variable preload tensions across samples. To test for a correlation between preload tension and supercontraction, 15 silk samples from each of two L. hesperus individuals were collected. These samples were mounted at different preload tensions, ranging from 0 to 170MPa. Supercontraction tests were then run as described above, and SS
Shrink=l0–l1
Stress
l0
l1
Low humidity
Stress
Three pictures were taken of each sample using polarized light microscopy at 1000⫻ (Blackledge et al., 2005a). Each strand diameter was measured using ImageJ (http://rsb.info.nih.gov/ij/) and the total cross-sectional area calculated. Two different aspects of supercontraction were measured: the stress generated in restrained fibers and the degree to which unrestrained silk shrank when exposed to water. Previous studies measured supercontraction as the degree of fiber shrinking (e.g. Work, 1981). However, the stress generated during supercontraction by the fiber is also important, as it will affect the performance of structures made of silk (webs or potentially, man-made silk structures). Supercontraction tests were carried out on a Nano Bionix tensile tester (MTS Corp., Oakridge, TN, USA) equipped with a humidity chamber, as described in Agnarsson et al. (Agnarsson et al., 2009a). The relative humidity inside the chamber could be set to any value between ~1% and ~95%. Silk samples were mounted at room humidity (5–15%) and pulled on at 0.1% strain, until just taut (Savage et al., 2004). Following the terminology adopted by Blackledge et al. (Blackledge et al., 2009a), the tests performed were WS0.1% tests (strained at 0.1% then wetted). Fig.1 is a diagram of the supercontraction test. Humidity was ramped up from ambient humidity to over 75% within 2min. When supercontraction critical humidity was reached, the hydrogen bonds were disrupted, freeing the molecules to move to lower energy states. However, the fiber was unable to shrink because it was held by the grips. Thus, stress instead developed within the fiber. We refer to this as supercontraction stress (SS) and calculated it using engineering stress as:
High humidity (>70%)
Supercontraction
Time Fig.1. The method used to measure supercontraction stress and percentage of shrink. The upper part of the figure illustrates the tensile testing device; the lower part is a typical curve of stress through time. A silk thread of length l0 (black line) is mounted between the grips of a tensile tester (grey rectangles) at low humidity and 0.1% strain. As the humidity rises to ~70%, silk supercontracts but the thread is held at constant length, which results in supercontraction stress. The thread is relaxed, at which point the stress goes back to zero. The thread is then slowly extended. Once the thread length passes the post-supercontraction length l1, stress rises again. Supercontraction percentage of shrink is calculated as the difference between the original length l0 and the final length l1.
and PS were recorded. For each individual, SS and PS were regressed versus preload tension. Spider phylogeny
Phylogenetic relationships may influence supercontraction of silk. For instance, the level of supercontraction of silk from closely related taxa may be more similar than that of distant taxa simply because of phylogenetic inertia. Independent contrasts (IC) were used to correct for the non-independence of related species (see Statistical analysis). No existing phylogeny includes all of the species in our study, but we estimated species relationships using Coddington’s Araneae phylogeny (Coddington, 2005) with additions from Raven (Raven, 1985) for tarantulas and Blackledge et al. (Blackledge et al., 2009b) for apical relationships within Orbiculariae (Fig.2). Web ecology and silk proteins
This study tried to relate supercontraction to the spinning of orb webs and the presence of MaSp2 silk proteins. Table1 describes the type of webs spun by each taxon and the presence or absence of MaSp2 in the silk of each taxon. The presence or absence of MaSp2 in silk was inferred from cDNA data from Garb et al. and Gatesy et al. (Garb et al., 2007; Gatesy et al., 2001) for mygalomorphs; Tian et al. (Tian et al., 2004) for Kukulcania sp.; Gatesy et al., Pouchkina-Stantcheva and McQueen-Mason, Rising et al. and Tian et al. (Gatesy et al., 2001; Pouchkina-Stantcheva and McQueen-Mason, 2004; Rising et al., 2007; Tian et al., 2004) for RTA clade species (Hololena, Amaurobius, Hogna, Dolomedes and Tengella); Gatesy et al. (Gatesy et al., 2001) for Tetragnathidae; Hinman and Lewis, Sponner et al. and Xu and Lewis (Hinman and Lewis, 1992; Sponner et al., 2005; Xu and Lewis, 1990) for Nephilidae; Gatesy et al. and Guerette et al. (Gatesy et al., 2001; Guerette et al., 1996) for Araneidae; and Ayoub et al., Ayoub and Hayashi and Gatesy et al. (Ayoub et al., 2007; Ayoub and Hayashi,
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Aphonopelma seemani Ephebopus uatuman Grammostola rosea Hypochilus thorelli Kukulcania hibernalis Pholcus phalangioides Haplogyne Diguetia canities Scytodes sp. Eresus kollari Salticus scenicus Hololena adnexa Amaurobius ferox Tengella radiata RTA clade Peucetia viridans Dolomedes tenebrosus Hogna helluo Uloborus diversus Pityohyphantes costatus Tetragnatha sp. Latrodectus hesperus Achaearanea tepidariorum Orbiculariae Synotaxus sp. Nephilidae Nephila clavipes Zygiella x-notata Araneus diadematus Verrucosa arenata Araneidae Larinioides sclopetarius Nuctenea umbratica
Mygalomorphae
Fig.2. Phylogeny of the taxa used in this study. Major clades are indicated by different colors.
Supercontraction stress (SS, MPa) Percentage of shrink (PS, %)
Evolution of silk supercontraction 16
3509
A
14 12 10 8 6 4 2 0 120
B
100 80 60 40 20 0
0
20
40
60 80 100 120 140 160 180 Preload tension (MPa)
2008; Gatesy et al., 2001) for Theridiidae. For RTA clade spiders, Rising et al. (Rising et al., 2007) suggested the presence of a protein somewhat similar to MaSp2, but much poorer in GPGXX motifs. Additionally, Gatesy et al. (Gatesy et al., 2001) did not find any MaSp2-like sequence in RTA clade spiders. Therefore, we considered the taxa from the RTA clade to lack MaSp2. The silk proteins of many of the taxa used here have not yet been characterized. In this case, the phylogeny was used to infer whether their silk likely contained MaSp2. MaSp2 is known in several Orbiculariae, including Nephilidae, Uloboridae and Araneidae, but is not found in the RTA clade. RTA clade spiders and Orbiculariae are all higher Entelegyne sensu Coddington and Levi (Coddington and Levi, 1991). Therefore, we considered all RTA clade spiders and all sister taxa to the higher Entelegyne to be lacking MaSp2, and all taxa derived from the RTA clade to have MaSp2. Among the taxa that possess MaSp2, the proportion of MaSp2 in the silk may affect silk properties and behavior (Liu et al., 2008b; Savage and Gosline, 2008a). However, data on the percentage of MaSp2 in the silk of various species are generally lacking. Therefore, we only used presence or absence of MaSp2 as a criterion in this study.
Fig.3. The relationship between preload tension and (A) supercontraction stress or (B) percentage of shrink. Silk from two Latrodectus individuals was tested; the blue squares and regression line represent the silk of the first individual and the red diamonds and regression line that of the second.
Statistical analysis
Evolution of supercontraction in spiders in relation to protein composition and web type
The average SS and PS per species were used in all the analyses. The analyses compared supercontraction between species with or without MaSp2 in their silk, and between species that spin or do not spin orb webs. A series of standard ANOVAs, with either SS or PS as the dependent variable, and either presence of MaSp2 or type of web (orb-web vs non-orb-web or no web) as the independent variable, were conducted. When testing the effect of web type, analyses were conducted both with all taxa and only within Orbiculariae species. The non-independence of phylogenetically related taxa was accounted for by following Garland et al.’s independent contrasts method (Garland et al., 1993). Using PDSIMUL and PDANOVA from the PDAP package; F distributions were created, taking into account the phylogeny and assuming no relation between SS or PS and presence of MaSp2 or web type. ANOVAs were run using PDSINGLE, with either SS or PS as the dependent variable, and either presence of
MaSp2 or type of web (orb-web vs non-orb-web or no web) as the independent variable. The F values from the ANOVA were compared with the critical values obtained from the simulated F distributions. Hogna helluo was removed from our data set for SS since stress data could not be collected for this species. RESULTS Correlation between SS, PS and preload tension
Supercontraction stress was strongly correlated with preload tension in the silk from both individuals of L. hesperus tested (linear regression: first individual, P0.0206; second individual, P
